Use of il-20 antagonists for treating pancreatic cancer

ABSTRACT

Methods for treating pancreatic cancer using an IL-20 antagonist, which can be an antibody that binds IL-20 or an IL-20 receptor, thereby blocking the signaling pathway mediated by IL-20.

BACKGROUND OF THE INVENTION

Pancreatic cancer is among the most lethal human cancers, in partbecause it typically spreads rapidly and is insensitive to manychemotherapeutic drugs. Pancreatic cancer, even when diagnosed in itsearly stages, often has a poor prognosis. Surgical removal of thepancreas remains the only curative option for treating pancreaticcancer. However, pancreatic cancer symptoms typically do not appearuntil advanced stages when the cancer has metastasized, therebyrendering surgical removal of the pancreas ineffective.

Interleukin IL-20 (IL-20) is a member of the IL-10 family, whichincludes IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26. Blumberg, et al.,2001, Cell 104:9-19; Pestka et al., 2004, Annu Rev Immunol 22:929-979.IL-20 is expressed in monocytes, epithelial cells, and endothelial cellsand acts on multiple cell types by activating a heterodimer receptorcomplex of either IL-20R1/IL-20R2 or IL-22R1/IL-20R2. Dumoutier, et al.,2001, J Immunol 167:3545-3549). IL-20 was found to be involved invarious inflammatory diseases, such as psoriasis (Blumberg et al., 2001;Sa et al., 2007, J Immunol 178:2229-2240; and Wei et al., 2005, ClinImmunol 117:65-72), rheumatoid arthritis (Hsu, et al., 2006, ArthritisRheum 54:2722-2733), atherosclerosis (Caligiuri, et al. 2006,Arterioscler Thromb Vasc Biol 26:1929-1930; and Chen et al., 2006,Arterioscler Thromb Vasc Biol 26:2090-2095), ischemic stroke (Chen etal., 2009, J Immunol 182:5003-5012), and renal failure (Li et al., 2008,Genes Immun 9:395-404). See also Wei et al., 2006, J Biomed Sci13:601-612.

SUMMARY OF THE INVENTION

The present disclosure is based on the unexpected results that IL-20might be involved in the pathogenesis of pancreatic cancer andantibodies binding to human IL-20 successfully inhibited tumor growthand prolonged survival rates in mice suffering from pancreatic cancervia blockade of the signaling pathway mediated by IL-20.

Accordingly, one aspect of the present disclosure relates to a methodfor treating pancreatic cancer or delaying the onset of pancreaticcancer in a subject, comprising administering to a subject in need ofthe treatment an effective amount of a pharmaceutical compositioncomprising an IL-20 antagonist. In some embodiments, the IL-20antagonist cam be an antibody that binds to IL-20 or an IL-20 receptor,thereby inhibiting a signaling pathway mediated by IL-20. For example,such an antibody may bind to an IL-20 protein (e.g., human IL-20) or maybind to an IL-20 receptor (e.g., a human IL-20 receptor such as R1subunit of an IL-20). Any of the antibodies used in the method describedherein can be a full-length antibody or an antigen-binding fragmentthereof. Alternatively, the antibody can be a human antibody, ahumanized antibody, a chimeric antibody, or a single-chain antibody.

When an antibody that binds human IL-20 is used in the method describedherein, it can be the monoclonal antibody mAb7E, an antigen-bindingfragment thereof, or a functional variant thereof. In one example, afunctional variant of mAb7E comprises the same complementary determiningregions (CDRs) as mAb7E. In another example, the functional variant is ahumanized antibody of mAb7E. Such a humanized antibody can comprises aheavy chain variable region (V_(H)), which comprises the amino acidsequence of SEQ ID NO:8, and a light chain variable region (V_(L)),which comprises the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:13.

Alternatively, an antibody that binds a human IL-20 receptor, e.g.,binds the IL-20R1 subunit, the IL-20R2 subunit, the IL-20R1/R2 complex,the IL-22R1 subunit, or the IL-22R1/IL-20R2 complex, can be used in themethod described herein. In some embodiments, the antibody binds subunitR1 of human IL-20 receptor. Such an antibody can be a full-lengthantibody or an antigen-binding fragment thereof. It also can be a humanantibody, a humanized antibody, a chimeric antibody, or a single-chainantibody. In one example, the antibody that binds subunit R1 of thehuman IL-20 receptor is an antibody comprising the same V_(H) and V_(L)as monoclonal antibody mAb51D or mAb7GW, or a functional variant ofmAb51D or mAb7GW. A functional variant can comprise the samecomplementary determining regions (CDRs) as mAb51D or mAb7GW.Alternatively, a functional variant can be a humanized antibody ofmAb51D or mAb7GW.

The subject to be treated in the method described herein (e.g., a methodin which an antibody that inhibits the IL-20 signaling pathway is used)can be a patient (e.g., a human patient) who has or is suspected ofhaving pancreatic cancer, which can be pancreatic adenocarcinoma ornon-adenocarcinoma. In some examples, the subject is a human patient whohas or is suspected of having pancreatic cancer.

Also within the scope of this disclosure are (a) pharmaceuticalcompositions for use in treating pancreatic cancer or delaying the onsetof pancreatic cancer in a subject, the pharmaceutical compositioncomprising one or more of the IL-20 antagonists described herein (e.g.,an antibody that inhibits the IL-20 signaling pathway such as anantibody that binds human IL-20 or human IL-20 receptor (R1, R2, or acomplex thereof); and (b) uses of the just-described pharmaceuticalcomposition in manufacturing a medicament for treating or delaying theonset of pancreatic cancer.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the inhibitory effect of mAb7E on tumor growthin mice having BxPC-3-induced pancreatic cancer. *: mIgG versus 7E,p<0.05. **: mIgG versus 7E, p<0.01

FIG. 2 shows the inhibitory effects of mAb7E on tumor weight. Panel A:an image showing tumors harvested from mice treated with PBS, mouseimmunoglobulin G, or mAb7E. Panel B: a chart showing tumor weight (mg)from mice treated with PBS, mouse immunoglobulin G, or mAb7E. **: mIgGversus 7E, p<0.01

FIG. 3 is a chart showing that treatment with mAb7E prolongs survivalrates in mice having KPC-induced pancreatic cancer. *: mIgG versus 7E,p<0.01.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence encoding the heavy chain variableregion of monoclonal antibody mAb7E.

SEQ ID NO:2 is the amino acid sequence of the heavy chain variableregion of monoclonal antibody mAb7E.

SEQ ID NO:3 is the nucleotide sequence encoding the light chain variableregion of monoclonal antibody mAb7E.

SEQ ID NO:4 is the amino acid sequence of the light chain variableregion of monoclonal antibody mAb7E.

SEQ ID NO:5 is the nucleotide sequence encoding the heavy chain variableregion of humanized antibodies HL1 and HL2 derived from mAb7E (precursorform, which includes a signal peptide).

SEQ ID NO:6 is the amino acid sequence of the heavy chain variableregion of humanized antibodies HL1 and HL2 derived from mAb7E (precursorform, which includes a signal peptide).

SEQ ID NO:7 is the nucleotide sequence encoding the heavy chain variableregion of humanized antibodies HL1 and HL2 derived from mAb7E (matureform, lacking the signal peptide).

SEQ ID NO:8 is the amino acid sequence of the heavy chain variableregion of humanized antibodies HL1 and HL2 derived from mAb7E (matureform, lacing the signal peptide).

SEQ ID NO:9 is the nucleotide sequence encoding the light chain variableregion of humanized antibody HL2 (precursor form, which includes asignal peptide).

SEQ ID NO:10 is the amino acid sequence of the light chain variableregion of humanized antibody HL2 (precursor form, which includes asignal peptide).

SEQ ID NO:11 is the nucleotide sequence encoding the light chainvariable region of humanized antibody HL2 (mature form, lacking thesignal peptide).

SEQ ID NO:12 is the amino acid sequence of the light chain variableregion of humanized antibody HL2 (mature form, lacking the signalpeptide).

SEQ ID NO:13 is the amino acid sequence of the light chain variableregion of humanized antibody HL1 (mature form, lacking the signalpeptide).

SEQ ID NO:14 is the amino acid sequence of the heavy chain of monoclonalantibody mAb7GW.

SEQ ID NO:15 is the nucleotide sequence encoding the heavy chain ofmonoclonal antibody mAb7GW.

SEQ ID NO:16 is the amino acid sequence of the light chain of monoclonalantibody mAb7GW.

SEQ ID NO:17 is the nucleotide sequence encoding the light chain ofmonoclonal antibody mAb7GW.

SEQ ID NO:18 is the amino acid sequence of the heavy chain of monoclonalantibody mAb51D.

SEQ ID NO:19 is the nucleotide sequence encoding the heavy chain ofmonoclonal antibody mAb51D.

SEQ ID NO:20 is the amino acid sequence of the light chain of monoclonalantibody mAb51D.

SEQ ID NO:21 is the nucleotide sequence encoding the light chain ofmonoclonal antibody mAb51D.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure reports the unexpected results that antibodiescapable of interfering with the IL-20 signaling pathway (e.g.,anti-IL-20 antibody such as mAb7E) successfully inhibited tumor growthand prolonged survival rates in a well-established pancreatic cancermouse model (the KPC mice). Accordingly, the present disclosure relatesto methods of treating pancreatic cancer (e.g., alleviating pancreaticcancer or delaying the onset of pancreatic cancer) in a subject using aneffective amount of an IL-20 antagonist, which can be an antibodycapable of interfering with the IL-20 signaling pathway.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

IL-20 Antagonists and Pharmaceutical Compositions Comprising Such

IL-20 is a pro-inflammatory cytokine that belongs to the IL-10 cytokinefamily. The IL-20 described herein refers to interleukin-20 and variantsthereof that retain at least part of the activity of IL-20. As usedherein, IL-20 includes all mammalian species of native sequence IL-20,including human, canine, feline, equine, or bovine. In one example, theIL-20 is a human IL-20 (GenBank accession no. NP_061194.2).

IL-20 activates the IL-20 signaling pathway via binding to IL-20receptor, which is a dimeric complex contains subunits IL-20R1 andIL-20R2 (also known as RA and RB). Such an IL-20 receptor is shared bythree functionally different cytokines, i.e., IL-19, IL-20, and IL-24,suggesting that this receptor mediates different signaling pathwaysdependent upon its binding to a specific cytokine. IL-20 is also capableof binding to a dimeric complex containing IL-20R2 and IL-22R1. TheIL-20 receptor disclosed herein refers to one or more polypeptides thatare capable of binding to and being activated by IL-20. IL-20 receptorsdisclosed herein include IL-20R1, IL-20R2 and IL-22R1 of any mammalianspecies, including, but are not limited to, human, canine, feline,equine, primate, or bovine. Examples of human IL-20 receptors includehIL-20R1 (GenBank Accession No. NM_014432.2), hIL-20R2 (GenBankAccession No. NM_144717.2) and hIL-22R1 (NM_181309.1). Sequences ofhuman IL-20 receptors have been described; for example, in U.S. Pat.Nos. 6,610,286; 7,122,632; 7,393,684; and 7,537,761; and U.S. Pat. App.Pub. Nos. 2006/0263850 A1; 2006/0263851 A1; 2008/0247945 A1, and2009/0074661 A1.

The IL-20 antagonist to be used in the methods described herein is amolecule that blocks, suppresses, or reduces (including significantly)the biological activity of IL-20, including downstream pathways mediatedby IL-20 signaling, such as receptor binding and/or elicitation of acellular response to IL-20. See US2011/0064731, which is incorporated byreference herein in its entirety. The term “antagonist” implies nospecific mechanism of biological action whatsoever, and is deemed toexpressly include and encompass all possible pharmacological,physiological, and biochemical interactions with IL-20 whether direct orindirect. For purpose of the present disclosure, it will be explicitlyunderstood that the term “antagonist” encompass all the previouslyidentified terms, titles, and functional states and characteristicswhereby the IL-20 itself (e.g., human IL-20), an IL-20 biologicalactivity (including but not limited to its ability to mediate any aspectof pancreatic cancer), or the consequences of the biological activity,are substantially nullified, decreased, or neutralized in any meaningfuldegree, e.g., by at least 20%, 50%, 70%, 85%, 90%, 100%, 150%, 200%,300%, or 500%, or by 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, or10⁴-fold.

Exemplary IL-20 antagonists include, but are not limited to, ananti-IL-20 antibody, an anti-sense nucleic acid molecule directed to anIL-20 (including an anti-sense nucleic acid directed to a nucleic acidencoding IL-20), a small interfering RNA (siRNA) directed toward anIL-20 nucleic acid, a microRNA directed toward an IL-20 nucleic acid, anIL-20 inhibitory compound, an anti-IL-20R antibody (e.g., an antibodyspecifically binds IL-20R1, IL-20R2, or the dimeric complex formedthereby), an antisense nucleic acid molecule directed to a subunit of anIL-20 receptor, an siRNA or a microRNA directed to a nucleic acidencoding a subunit of an IL-20 receptor, or an IL-20R inhibitorycompound. In some embodiments, an IL-20 antagonist binds IL-20 or IL-20receptor and prevents the formation of IL-20-IL-20R complex, therebyinhibiting the IL-20 signaling pathway. In other embodiments, an IL-20antagonist inhibits or reduces IL-20 synthesis and/or production(release). Such antagonists include antisense molecules, siRNAs andmicroRNAs.

Antibodies Capable of Interfering with the IL-20 Signaling Pathway

An antibody (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” encompassesnot only intact (i.e., full-length) polyclonal or monoclonal antibodies,but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)₂,Fv), single chain (scFv), mutants thereof, fusion proteins comprising anantibody portion, humanized antibodies, chimeric antibodies, diabodies,linear antibodies, single chain antibodies, multispecific antibodies(e.g., bispecific antibodies) and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. An antibody includes an antibody of any class, suchas IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class. Depending on the antibody aminoacid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The antibodies to be used in the methods described herein can be murine,rat, human, or any other origin (including chimeric or humanizedantibodies). In some examples, the antibody comprises a modifiedconstant region, such as a constant region that is immunologicallyinert, e.g., does not trigger complement mediated lysis, or does notstimulate antibody-dependent cell mediated cytotoxicity (ADCC). ADCCactivity can be assessed using methods disclosed in U.S. Pat. No.5,500,362. In other embodiments, the constant region is modified asdescribed in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No.PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.

Any of the antibodies described herein can be either monoclonal orpolyclonal. A “monoclonal antibody” refers to a homogenous antibodypopulation and a “polyclonal antibody” refers to a heterogenous antibodypopulation. These two terms do not limit the source of an antibody orthe manner in which it is made.

In one example, the antibody used in the methods described herein is ahumanized antibody. Humanized antibodies refer to forms of non-human(e.g., murine) antibodies that are specific chimeric immunoglobulins,immunoglobulin chains, or antigen-binding fragments thereof that containminimal sequence derived from non-human immunoglobulin. For the mostpart, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs “derived from” one or more CDRs from theoriginal antibody. Humanized antibodies may also involve affinitymaturation.

In another example, the antibody described herein is a chimericantibody, which can include a heavy constant region and a light constantregion from a human antibody. Chimeric antibodies refer to antibodieshaving a variable region or part of variable region from a first speciesand a constant region from a second species. Typically, in thesechimeric antibodies, the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals (e.g., a non-human mammal such as mouse, rabbit, and rat), whilethe constant portions are homologous to the sequences in antibodiesderived from another mammal such as human. In some embodiments, aminoacid modifications can be made in the variable region and/or theconstant region.

In some examples, the antibody disclosed herein specifically binds atarget antigen, such as human IL-20 or one of the two subunits of ahuman IL-20 receptor (e.g., IL-20R1). An antibody that “specificallybinds” (used interchangeably herein) to a target or an epitope is a termwell understood in the art, and methods to determine such specificbinding are also well known in the art. A molecule is said to exhibit“specific binding” if it reacts or associates more frequently, morerapidly, with greater duration and/or with greater affinity with aparticular target antigen than it does with alternative targets. Anantibody “specifically binds” to a target antigen if it binds withgreater affinity, avidity, more readily, and/or with greater durationthan it binds to other substances. For example, an antibody thatspecifically (or preferentially) binds to an IL-20 epitope is anantibody that binds this IL-20 epitope with greater affinity, avidity,more readily, and/or with greater duration than it binds to other IL-20epitopes or non-IL-20 epitopes. It is also understood by reading thisdefinition that, for example, an antibody that specifically binds to afirst target antigen may or may not specifically or preferentially bindto a second target antigen. As such, “specific binding” or “preferentialbinding” does not necessarily require (although it can include)exclusive binding. Generally, but not necessarily, reference to bindingmeans preferential binding.

Antibodies capable of interfering with the IL-20 signaling pathway canbe an antibody that binds an IL-20 (e.g., a human IL-20) and inhibitsIL-20 biological activity and/or downstream pathways mediated by IL-20.Alternatively, such antibodies can be antibodies that bind an IL-20receptor (IL-20R), e.g., bind to one or both of the subunits of theIL-20 receptor, and suppress the downstream signaling pathways mediatedby the receptor triggered by IL-20.

(i) Anti-IL-20 Antibodies

An anti-IL-20 antibody is an antibody capable of binding to IL-20 andinhibits IL-20 biological activity and/or downstream pathway(s) mediatedby IL-20 signaling. In some examples, an anti-IL-20 antibody used in themethods described herein suppresses the IL-20 signaling pathway by atleast 20%, at least 40%, at least 50%, at least 75%, at least 90%, atleast 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, at least 100-fold, or at least1000-fold. Examples of anti-IL-20 antibodies include, but are notlimited to, those disclosed in U.S. Pat. Nos. 7,435,800; 7,115,714;7,119,175; 7,151,166; and 7,393,684; and PCT publications WO2007/081465; WO 99/27103; WO 2004/085475; and WO 2005052000.

The binding affinity of an anti-IL-20 antibody to IL-20 (such as humanIL-20) can be less than any of about 100 nM, about 50 nM, about 10 nM,about 1 nM, about 500 pM, about 100 pM, or about 50 pM to any of about 2pM. Binding affinity can be expressed K_(D) or dissociation constant,and an increased binding affinity corresponds to a decreased K_(D). Oneway of determining binding affinity of antibodies to IL-20 is bymeasuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of an anti-IL-20 Fab fragment of an antibody can be determinedby surface plasmon resonance (BIAcore3000™ surface plasmon resonance(SPR) system, BIAcore, INC, Piscaway N.J.). Kinetic association rates(k_(on)) and dissociation rates (k_(off)) (generally measured at 25° C.)are obtained; and equilibrium dissociation constant (K_(D)) values arecalculated as k_(off)/k_(on).

In some embodiments, the antibody binds human IL-20, and does notsignificantly bind an IL-20 from another mammalian species. In someembodiments, the antibody binds human IL-20 as well as one or more IL-20from another mammalian species. In still other embodiments, the antibodybinds IL-20 and does not significantly cross-react with other cytokines(such as the related cytokines IL-10, IL-17A, IL-19, IL-22, IL-24 andIL-26). The epitope(s) bound by the antibody can be continuous ordiscontinuous.

In some embodiments, the anti-IL-20 antibody described herein isanti-IL-20 antibody 7E, which refers to monoclonal antibody mAb 7E andits functional variants. MAb 7E is produced by the hybridoma cell linedeposited at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, U.S.A. and assigned a depositnumber PTA-8687. This hybridoma cell line will be released to the publicirrevocably and without restriction/condition upon granting a US patenton this application, and will be maintained in the ATCC for a period ofat least 30 years from the date of the deposit for the enforceable lifeof the patent or for a period of 5 years after the date of the mostrecent. See also U.S. Pat. Nos. 8,206,712 and 7,611,705, the relevantdisclosures of each of which are incorporated by reference herein.

The amino acid sequences and encoding nucleotide sequences of the heavychain variable region (V_(H)) and light chain variable region (V_(L)) ofmAb7E are produced below:

Nucleotide sequence (SEQ ID NO: 1) and amino acid sequence(SEQ ID NO: 2) of mAb 7E heavy chain variable regiongaa ttg aag ctt gag gag tct gga gga ggc ttg gtg cag cct gga 45 E   L   K   L   E   E   S   G   G   G   L   V   Q   P   G 15gga tcc atg aaa ctc tct tgt gct gcc tct gga ttc act ttt agt 90 G   S   M   K   L   S   C   A   A   S   G   F   T   F   S 30gac gcc tgg atg gac tgg gtc cgc cag tct cca gag aag ggg ctt 135 D   A   W   M   D   W   V   R   Q   S   P   E   K   G   L 45gag tgg att gct gaa att aga agc aaa gct aat aat tat gca aca 180 E   W   I   A   E   I   R   S   K   A   N   N   Y   A   T 60tac ttt gct gag tct gtg aaa ggg agg ttc acc atc tca aga gat 215 Y   F   A   E   S   V   K   G   R   F   T   I   S   R   D 75gat tcc aaa agt ggt gtc tac ctg caa atg aac aac tta aga gct 270 D   S   K   S   G   V   Y   L   Q   M   N   N   L   R   A 90gag gac act ggc att tat ttc tgt acc aag tta tca cta cgt tac 315 E   D   T   G   I   Y   F   C   T   K   L   S   L   R   Y 105tgg ttc ttc gat gtc tgg ggc gca ggg acc acg gtc acc gtc tcc 360 W   F   F   D   V   W   G   A   G   T   T   V   T   V   S 120 tca 363 S 121 Nucleotide sequence (SEQ ID NO: 3) and amino acid sequence(SEQ ID NO: 4) of mAb 7E light chain variable regiongat ttt gtg atg acc cag act cca ctc act ttg tcg gtt acc att 45 D   F   V   M   T   Q   T   P   L   T   L   S   V   T   I 15gga caa cca gcc tcc atc tct tgc aag tca agt cag agc ctc ttg 90 G   Q   P   A   S   I   S   C   K   S   S   Q   S   L   L 30gat agt gat gga aag aca tat ttg aat tgg ttg tta cag agg cca 135 D   S   D   G   K   T   Y   L   N   W   L   L   Q   R   P 45ggc cag tct cca aag cac ctc atc tat ctg gtg tct aaa ctg gac 180 G   Q   S   P   K   H   L   I   Y   L   V   S   K   L   D 60tct gga gtc cct gac agg ttc act ggc agt gga tca ggg acc gat 215 S   G   V   P   D   R   F   T   G   S   G   S   G   T   D 75ttc aca ctg aga atc agc aga gtg gag gct gag gat ttg gga gtt 270 F   T   L   R   I   S   R   V   E   A   E   D   L   G   V 90tat tat tgc tgg caa agt aca cat ttt ccg tgg acg ttc ggt gga 315 Y   Y   C   W   Q   S   T   H   F   P   W   T   F   G   G 105ggc acc aag ctg gaa atc aaa cgg 339  G   T   K   L   E   I   K   R 113

A functional variant (equivalent) of mAb7E has essentially the sameepitope-binding specificity as mAb7E and exhibits at least 20% (e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the activity ofneutralizing a signaling pathway mediated by IL-20 as relative to mAb7E.In some embodiments, a functional variant of mAb7E contains the sameregions/residues responsible for antigen-binding as mAb7E, such as thesame specificity-determining residues in the CDRs or the whole CDRs. Theregions/residues that are responsible for antigen-binding can beidentified from amino acid sequences of the heavy chain/light chainsequences of mAb7GW or mAb51D (shown above) by methods known in the art.See, e.g., www.bioinf.org.uk/abs; Almagro, J. Mol. Recognit. 17:132-143(2004); and Chothia et al., J. Mol. Biol. 227:799-817 (1987).

In addition, determination of CDR regions in an antibody is well withinthe skill of the art. There are at least two techniques for determiningCDRs: (1) an approach based on cross-species sequence variability (i.e.,Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed.,1991, National Institutes of Health, Bethesda Md.)); and (2) an approachbased on crystallographic studies of antigen-antibody complexes (Chothiaet al. (1989) Nature 342:877; Al-lazikani et al (1997) J. Molec. Biol.273:927-948)). As used herein, a CDR may refer to CDRs defined by eitherapproach or by a combination of both approaches.

In some examples, a functional variant of mAb7E comprises a V_(H) chainthat includes a V_(H) CDR1, V_(H) CDR2, and V_(H) CDR3 at least 75%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the corresponding V_(H)CDRs of mAb7E, and a V_(L) chain that includes a V_(L) CDR1, V_(L) CDR2,and V_(L) CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identicalto the corresponding V_(H) CDRs of mAb7E.

Alternatively, the functional variant of mAb7E comprises a V_(H) chainat least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the V_(H)chain (mature or precursor) of mAb7E and a V_(L) chain at least 75%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the V_(L) chain (matureof precursor) of mAb7E.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In other examples, a functional variant of mAb7E comprises a V_(H) chainthat includes up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid residuevariations in the V_(H) CDR regions (V_(H) CDR1, CDR2, and/or CDR3) ascompared to the V_(H) CDRs of mAb7E, and/or a V_(L) chain that includesup to 5 (e.g., 1, 2, 3, 4, or 5) amino acid residue variations in theV_(L) CDR regions (V_(L) CDR1, CDR2, and/or CDR3) as compared to theV_(H) CDRs of mAb7E.

Functional variants of mAb7E are also disclosed in U.S. Pat. No.7,611,705 and US2011/0064731, both of which are incorporated byreference herein.

In one example, a functional variant of mAb7E is a humanized antibodyderived from mAb7E. Provided below are exemplary humanized mAb7Eantibodies HL1 and HL2; see also U.S. Pat. No. 8,597,647, the relevantdisclosures therein are incorporated by reference.

Amino acid sequence and encoding nucleotide sequence of the V_(H) chainof humanized anti-IL-20 antibodies HL1 and HL2:

(SEQ ID NO: 5)                 ATG TAC TTG GGA CTG AAC TAT GTT(SEQ ID NO: 6)                  M   Y   L   G   L   N   Y   VTTC ATC GTT TTT CTC CTG AAT GGT GTC CAG AGT GAA F   I   V   F   L   L   N   G   V   Q   S   EGTG CAG CTT GTG GAG TCT GGA GGA GGC TTG GTG CAG V   Q   L   V   E   S   G   G   G   L   V   QCCT GGA GGA TCC CTG AAA CTC TCT TGT GCT GCC TCT P   G   G   S   L   K   L   S   C   A   A   SGGA TTC ACT TTT AGT GAC GCC TGG ATG GAC TGG GTC  G   F   T   F   S   

   

   

   

   

   W   V CGC CAG GCT TCC GGG AAG GGG CTT GAG TGG ATT GCT R   Q   A   S   G   K   G   L   E   W   I   AGAA ATT AGA AGC AAA GCT AAT AAT TAT GCA ACA TAC  

   

   

   

   

   

   

   

   

   

   

   

TTT GCT GAG TCT GTG AAA GGG AGG TTC ACC ATC TCA  

   

   

   

   

   

   

   R   F   T   I   S AGA GAT GAT TCC AAA AAC ACC GCC TAC CTG CAA ATG R   D   D   S   K   N   T   A   Y   L   Q   MAAC AGC TTA AAA ACC GAG GAC ACT GCC GTT TAT TAC N   S   L   K   T   E   D   T   A   V   Y   YTGT ACC AAG TTA TCA CTG CGT TAC TGG TTC TTC GAT  C   T   K  

   

   

   

   

   

   

   

   

GTC TGG GGC CAG GGG ACC CTG GTC ACC GTC TCC TCA  

  W   G   Q   G   T   L   V   T   V   S   S

The underlined region refers to the signal peptide and theboldfaced/italic regions are the CDRs. SEQ ID NOs: 8 and 7 represent themature V_(H) amino acid sequence (lacking the signal peptide) and itsencoding nucleotide sequence, respectively.

Amino acid sequence and encoding nucleotide sequence of the V_(L) chain(VL2) of a humanized anti-IL-20 antibody HL2:

(SEQ ID NO: 9)             ATG ATG AGT CCT GCC CAG TTC CTG TTT(SEQ ID NO: 10)              M   M   S   P   A   Q   F   L   FCTG TTG GTG CTC TGG ATT CGG GAA ACC AAC GGT GAT L   L   V   L   W   I   R   E   T   N   G   DATC GTG ATG ACC CAG ACT CCA CTC TCT TTG TCC GTT  

  V   M   T   Q   T   P   L   S   L   S   VACC CCT GGA CAA CCA GCC TCC ATC TCT TGC AAG TCA T   P   G   Q   P   A   S   I   S   C  

   

  AGT CAG AGC CTC TTG GAT AGT GAT GGA AAG ACA TAT   

   

   

   

   

   

   

   

   

   

   

   

TTG AAT TGG TTG TTA CAG AAG CCA GGC CAG TCT CCA  

   

  W   L   L   Q   K   P   G   Q   S   PCAG CAC CTC ATC TAT CTG GTG TCT AAA CTG GAC TCT  Q   H   L   I   Y  

   

   

   

   

   

   

GGA GTC CCT GAC AGG TTC AGT GGC AGT GGA TCA GGG G   V   P   D   R   F   S   G   S   G   S   GACC GAT TTC ACA CTG AAA ATC AGC AGA GTG GAG GCT T   D   F   T   L   K   I   S   R   V   E   AGAG GAT GTT GGA GTT TAT TAT TGC TGG CAA AGT ACA E   D   V   G   V   Y   Y   C  

   

   

   

CAT TTT CCC TGG ACC TTC GGT GGA GGC ACC AAG GTG  

   

   

   

   

   F   G   G   G   T   K   V GAA ATC AAA  E   I   K

The underlined region refers to the signal peptide and theboldfaced/italic regions are the CDRs. SEQ ID NOs: 12 and 11 representthe mature V_(L) amino acid sequence (lacking the signal peptide) andits encoding nucleotide sequence, respectively.

Humanized antibody HL1 comprises the same V_(H) chain as HL2 and a V_(L)chain (SEQ ID NO:13; mature form) that is otherwise identical to theV_(L) of HL2 except that the I residue at position 2 of mature V_(L) ofHL2 is replaced with F.

Also disclosed herein are functional variants of the above-notedhumanized antibodies HL1 and HL2. Such functional variants can comprisea V_(H) chain that comprises an amino acid sequence at least 85% (e.g.,90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to that of theV_(H) of HL1 and HL2 (precursor or mature form; SEQ ID NO:6 and SEQ IDNO:8, respectively) and a V_(L) chain that has an amino acid sequence atleast 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical tothat of the V_(L) of HL2 (precursor or mature form; SEQ ID NO:10 and SEQID NO:12, respectively). These variants are capable of binding to anIL-20 molecule, particularly a human IL-20 molecule. In some examples,the variants possess similar antigen-binding affinity relative to theexemplary humanized antibody described above (e.g., having aK_(d)<4×10).

(b) Anti-IL-20R Antibodies

An anti-IL-20R antibody is an antibody capable of binding to an IL-20R(e.g., binding to either one of its two subunits or binding to thedimeric complex) and inhibits the biological activity of the IL-20Rand/or its downstream pathway(s) mediated by IL-20. In some examples, ananti-IL-20 antibody used in the methods described herein suppresses theIL-20 signaling pathway by at least 20%, at least 40%, at least 50%, atleast 75%, at least 90%, at least 100%, or by at least 2-fold, at least5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least100-fold, or at least 1000-fold. In some examples, the anti-IL-20Rantibody specifically binds IL-20R1, such as human IL-20R1. Such anantibody may have low affinity to IL-20R2 or the IL-20R1/IL-20R2 complexor does not bind IL-20R2 or the IL-20R1/IL-20R2 complex. In otherexamples, the anti-IL-20R antibody specifically binds IL-20R2, such ashuman IL-20R2. Such an antibody may have low affinity to IL-20R1 or theIL-20R1/IL-20R2 complex or does not bind IL-20R1 or the IL-20R1/IL-20R2complex. In yet other examples, the anti-IL-20R antibody describedherein specifically binds the IL-20R1/IL-20R2 complex.

The binding affinity of an anti-IL-20R antibody to IL-20R or a subunitthereof (such as human IL-20R or human IL-20R1) can be less than any ofabout 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about100 pM, or about 50 pM to any of about 2 pM. Binding affinity can beexpressed K_(D) or dissociation constant, and an increased bindingaffinity corresponds to a decreased K_(D). One way of determiningbinding affinity of antibodies to IL-20R is by measuring bindingaffinity of monofunctional Fab fragments of the antibody. To obtainmonofunctional Fab fragments, an antibody (for example, IgG) can becleaved with papain or expressed recombinantly. The affinity of ananti-IL-20R Fab fragment of an antibody can be determined by surfaceplasmon resonance (BIAcore3000™ surface plasmon resonance (SPR) system,BIAcore, INC, Piscaway N.J.). Kinetic association rates (k_(on)) anddissociation rates (k_(off)) (generally measured at 25° C.) areobtained; and equilibrium dissociation constant (K_(D)) values arecalculated as k_(off)/k_(on).

In some embodiments, the antibody binds human IL-20R or a subunitthereof (e.g., human IL-20R1), and does not significantly bind an IL-20Rfrom another mammalian species. In some embodiments, the antibody bindshuman IL-20R as well as one or more IL-20R from another mammalianspecies. In still other embodiments, the antibody binds IL-20R and doesnot significantly cross-react with other cytokine receptors. Theepitope(s) bound by the antibody can be continuous or discontinuous.

In some embodiments, the antibody used in the methods described hereinis an antibody having the same heavy chain and light chain variableregions (V_(H) and V_(L)) as those of monoclonal antibody mAb7GW ormAb51D, the monoclonal antibodies, an antigen-binding fragment thereof,or a functional equivalent of either mAb7GW or mAb51D. US2011/0256093,which is herein incorporated by reference in its entirety. Shown beloware the amino acid sequences of the heavy chains and light chains ofmAb7GW and mAb51D, as well as their encoding nucleotide sequences.

Heavy Chain of mAb7GW: Amino Acid Sequence (SEQ ID NO: 14)M R V L I L L W L F T A F P G I L S V V Q L Q E S G P G L V K P S Q S L S L T C T V T G      Signal peptide Y S I T  S D Y A W N  W I R Q F P G N R L E W M G Y I D Y S G S T K Y N P S L K S  R I S V         CDR1                                          CDR2T R D T S K N Q F F L Q L N S V T T E D T A T Y Y C A R  D F G D A Y W G Q G T L V T V S                                 CDR3A A K T T P P S V Y P L A P G S A A Q T N S M V T L G C L V K G Y F P E P V T V T W N SG S L S S G V H T F P A V L Q S D L Y T L S S S V T V P S S T W P S E T V T C N V A H PA S S T K V D K K I V P R D C G C K P C I C T V P E V S S V F I F P P K P K D V L T I TL T P K V T C V V V D I S K D D P E V Q F S W F V D D V E V H T A Q T Q P R E E Q F N ST F R S V S E L P I M H Q D W L N G K E F K C R V N S A A F P A P I E K T I S K T K G RP K A P Q V Y T I P P P K E Q M A K D K V S L T C M I T D F F P E D I T V E W Q W N G QP A E N Y K N T Q P I M D T D G S Y F V Y S K L N V Q K S N W E A G N T F T C S V L H E G L H N H H T E K S L S H S P G K(The italic region refers to the heavy chain constant region).Nucleotide Sequence (SEQ ID NO: 15)ATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCTGTTGTGCAGCTTCAGGAGTCGGGACCTGGCCTG     Signal peptideGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAAC TGGATCCGGCAG                                                             CDR1TTTCCAGGAAACAGACTGGAGTGGATGGGCTACATAGACTACAGTGGTAGCACTAAATACAACCCCTCTCTCAAAAGT CGAATCTCT                                              CDR2GTCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGACTTTGGTGATGCTTACTGGGGCCAGGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCC    CDR3CCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAAACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAAATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA(The italic region encodes the heavy chain constant region.)Light Chain of mAb7GW: Amino Acid Sequence (SEQ ID NO: 16)M D S Q A Q V L M L L L L W V S G S C G D I V M S Q S P S S L A V S V G E K V T M S C K        Signal peptide S S Q S L L Y S R N Q K N Y L A W Y Q L K P G Q S P K L L I Y  W A S T R E S  G V P D R F          CDR1                                                   CDR2T G S G S G T D F T L T I S S V K A E D L A V Y Y C  Q Q Y Y S Y P L T F G A G T K L E L                                                       CDR3K R A D A A P T V S I F P P S S E Q L T S G G A S V V C F L N N F Y P K D I N V K W K ID G S E R Q N G V L N S W T D Q D S K D S T Y S M S S T L T L T K D E Y E R H N S Y T CE A T H K T S T S P I V K S F N R N E C(The italic region refers to the light chain constant region.)Nucleotide Sequence (SEQ ID NO: 17)ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTTCCTGTGGGGACATTGTGATGTCACAGTCTCCATCC          Signal peptide TCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGGAATCAAAAGAACTAC                                                           CDR1 TTGGCCTGGTACCAGCTGAAGCCAGGGCAGTCTCCTAAACTGCTGATTTAC TGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGC                                                       CDR2TTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAA TATTATAGCTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACDR3CCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG(The italic region encodes the light chain constant region.)Heavy Chain of mAb51D: Amino Acid Sequence (SEQ ID NO: 18)MNFGLSLIFLALILKGVQCEVQLVEAGGDLVKPGGSLKLSCAASGFSLS NYGMS WVRQTPDKRLEWVASISSGGRFTSYPDSVRG RF     Signal peptide                               CDR1                    CDR2TISRDNAKNTLYLQMSGLKSEDTAMYYCAR HDGNGGDYWGQGTSVTVSSAKTTPPSVYPLALGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTSNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(The italic region refers to the heavy chain constant region.)Nucleotide Sequence (SEQ ID NO: 19)ATGAACTTCGGGCTCAGCCTGATTTTCCTTGCCCTCATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGGCTGGGGGAGAC          Signal peptideTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCGGCCTCTGGATTCAGTTTGAGTAACTATGGCATGTCC TGGGTTCGCCAG                                                                 CDR1ACTCCAGACAAGAGGCTGGAGTGGGTCGCAAGCATTAGTAGTGGTGGTCGTTTCACCTCCTATCCAGACAGTGTGAGGGGG CGATTC                                                 CDR2ACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCGGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGA CACGACGGCAACGGTGGGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTAT            CDR3CCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA(The italic region encodes the heavy chain constant region.)Light Chain of mAb51D: Amino Acid Sequence (SEQ ID NO: 20)MDFQVQIFSFLLISASVIMSRGQIVLSQFPAILSASPGEKVTMTC RARSSVSFMH WYQQKPGSSPKPWIYATSNLAS GVPPRFSGSG     Signal peptide                             CDR1                  CDR2SGTSYSLTISRVEAEDAATYYC QQWSSNPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGS                       CDR3ERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(The italic region refers to the light chain constant region)Nucleotide Sequence (SEQ ID NO: 21)ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCTTCAGTCATAATGTCCAGAGGACAAATTGTTCTCTCCCAGTTT                   Signal peptideCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGGTCAAGTGTAAGTTTCATGCAC TGGTACCAG                                                          CDR1CAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTAT GCCACATCCAACCTGGCTTCTGGAGTCCCTCCTCGCTTCAGTGGCAGTGGG                                            CDR2TCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCA                                                                           CDR3TACACGTTCGGAGGGGGGACTAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG(The italic region encodes the light chain constant region.)

A functional equivalent of mAb7GW or mAb51D has the same epitope-bindingspecificity as mAb7GW or mAb51D and exhibits at least 20% (e.g., 30%,40%, 50%, 60%, 70%, 80%, 90%, or greater) of the activity ofneutralizing a signaling pathway mediated by IL-20R1 as relative tomAb7GW or mAb51D. In some embodiments, a functional equivalent of mAb7GWor mAb51D contains the same regions/residues responsible forantigen-binding as mAb7GW or mAb51D, such as the samespecificity-determining residues in the CDRs or the whole CDRs. Theregions/residues that are responsible for antigen-binding can beidentified from amino acid sequences of the heavy chain/light chainsequences of mAb7GW or mAb51D (shown above) by methods known in the art.See, e.g., www.bioinf.org.uk/abs; Almagro, J. Mol. Recognit. 17:132-143(2004); and Chothia et al., J. Mol. Biol. 227:799-817 (1987).

In some examples, a functional equivalent (variant) of mAb7GW or mAb51Dcomprises a V_(H) chain that includes a V_(H) CDR1, V_(H) CDR2, andV_(H) CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical tothe corresponding V_(H) CDRs of mAb7GW or mAb51D, and a V_(L) chain thatincludes a V_(L) CDR1, V_(L) CDR2, and V_(L) CDR3 at least 75% (e.g.,80%, 85%, 90%, 95%, or 98%) identical to the corresponding V_(H) CDRs ofmAb7GW or mAb51D.

Alternatively, the functional equivalent of mAb7GW or mAb51D comprises aV_(H) chain at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical tothe V_(H) chain (mature or precursor) of mAb7GW or mAb51D and a V_(L)chain at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to theV_(L) chain (mature of precursor) of mAb7GW or mAb51D.

In other examples, a functional equivalent of mAb7GW or mAb51D comprisesa V_(H) chain that includes up to 5 (e.g., 1, 2, 3, 4, or 5) amino acidresidue variations in the V_(H) CDR regions (V_(H) CDR1, CDR2, and/orCDR3) as compared to the V_(H) CDRs of mAb7GW or mAb51D, and/or a V_(L)chain that includes up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid residuevariations in the V_(L) CDR regions (V_(L) CDR1, CDR2, and/or CDR3) ascompared to the V_(H) CDRs of mAb7GW or mAb51D.

(c) Antibody Preparation

Antibodies capable of interfering with the IL-20 signaling pathway asdescribed herein can be made by any method known in the art. See, forexample, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York.

In some embodiments, antibodies specific to a target antigen (e.g.,human IL-20 or IL-20R1) can be made by the conventional hybridomatechnology. The full-length target antigen or a fragment thereof,optionally coupled to a carrier protein such as KLH, can be used toimmunize a host animal for generating antibodies binding to thatantigen. The route and schedule of immunization of the host animal aregenerally in keeping with established and conventional techniques forantibody stimulation and production, as further described herein.General techniques for production of mouse, humanized, and humanantibodies are known in the art and are described herein. It iscontemplated that any mammalian subject including humans or antibodyproducing cells therefrom can be manipulated to serve as the basis forproduction of mammalian, including human hybridoma cell lines.Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the anti-IL-20 monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalimmunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, orfluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies capable of interfering with the IL-20 signalingpathway. Hybridomas that produce such antibodies may be grown in vitroor in vivo using known procedures. The monoclonal antibodies may beisolated from the culture media or body fluids, by conventionalimmunoglobulin purification procedures such as ammonium sulfateprecipitation, gel electrophoresis, dialysis, chromatography, andultrafiltration, if desired. Undesired activity if present, can beremoved, for example, by running the preparation over adsorbents made ofthe immunogen attached to a solid phase and eluting or releasing thedesired antibodies off the immunogen. Immunization of a host animal witha target antigen or a fragment containing the target amino acid sequenceconjugated to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N═C═NR,where R and R1 are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, an antibody (monoclonal or polyclonal) of interest (e.g.,produced by a hybridoma) may be sequenced and the polynucleotidesequence may then be cloned into a vector for expression or propagation.The sequence encoding the antibody of interest may be maintained invector in a host cell and the host cell can then be expanded and frozenfor future use. In an alternative, the polynucleotide sequence may beused for genetic manipulation to “humanize” the antibody or to improvethe affinity (affinity maturation), or other characteristics of theantibody. For example, the constant region may be engineered to moreresemble human constant regions to avoid immune response if the antibodyis used in clinical trials and treatments in humans. It may be desirableto genetically manipulate the antibody sequence to obtain greateraffinity to the target antigen and greater efficacy in inhibiting thesignaling pathway mediated by IL-20. It will be apparent to one of skillin the art that one or more polynucleotide changes can be made to theantibody and still maintain its binding specificity to the targetantigen.

In other embodiments, fully human antibodies can be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse® fromAmgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.). In another alternative, antibodies maybe made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; andWinter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, thephage display technology (McCafferty et al., (1990) Nature 348:552-553)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)2 fragments canbe produced by pepsin digestion of an antibody molecule, and Fabfragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments.

Genetically engineered antibodies, such as humanized antibodies,chimeric antibodies, single-chain antibodies, and bi-specificantibodies, can be produced via, e.g., conventional recombinanttechnology. In one example, DNA encoding a monoclonal antibodiesspecific to a target antigen can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoone or more expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese hamster ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. See, e.g., PCT Publication No. WO87/04462. The DNA can then be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison et al., (1984) Proc.Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, genetically engineeredantibodies, such as “chimeric” or “hybrid” antibodies; can be preparedthat have the binding specificity of a target antigen.

Techniques developed for the production of “chimeric antibodies” arewell known in the art. See, e.g., Morrison et al. (1984) Proc. Natl.Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; andTakeda et al. (1984) Nature 314:452.

Methods for constructing humanized antibodies are also well known in theart. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033(1989). In one example, variable regions of V_(H) and V_(L) of a parentnon-human antibody are subjected to three-dimensional molecular modelinganalysis following methods known in the art. Next, framework amino acidresidues predicted to be important for the formation of the correct CDRstructures are identified using the same molecular modeling analysis. Inparallel, human V_(H) and V_(L) chains having amino acid sequences thatare homologous to those of the parent non-human antibody are identifiedfrom any antibody gene database using the parent V_(H) and V_(L)sequences as search queries. Human V_(H) and V_(L) acceptor genes arethen selected.

The CDR regions within the selected human acceptor genes can be replacedwith the CDR regions from the parent non-human antibody or functionalvariants thereof. When necessary, residues within the framework regionsof the parent chain that are predicted to be important in interactingwith the CDR regions (see above description) can be used to substitutefor the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology bylinking a nucleotide sequence coding for a heavy chain variable regionand a nucleotide sequence coding for a light chain variable region.Preferably, a flexible linker is incorporated between the two variableregions. Alternatively, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can beadapted to produce a phage scFv library and scFv clones specific toIL-20R1 or IL-20R2 can be identified from the library following routineprocedures. Positive clones can be subjected to further screening toidentify those that suppress IL-20 receptor activity.

Antibodies obtained following a method known in the art and describedherein can be characterized using methods well known in the art. Forexample, one method is to identify the epitope to which the antigenbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan antibody binds. The epitope can be a linear epitope, i.e., containedin a single stretch of amino acids, or a conformational epitope formedby a three-dimensional interaction of amino acids that may notnecessarily be contained in a single stretch (primary structure linearsequence). Peptides of varying lengths (e.g., at least 4-6 amino acidslong) can be isolated or synthesized (e.g., recombinantly) and used forbinding assays with an antibody. In another example, the epitope towhich the antibody binds can be determined in a systematic screening byusing overlapping peptides derived from the target antigen sequence anddetermining binding by the antibody. According to the gene fragmentexpression assays, the open reading frame encoding the target antigen isfragmented either randomly or by specific genetic constructions and thereactivity of the expressed fragments of the antigen with the antibodyto be tested is determined. The gene fragments may, for example, beproduced by PCR and then transcribed and translated into protein invitro, in the presence of radioactive amino acids. The binding of theantibody to the radioactively labeled antigen fragments is thendetermined by immunoprecipitation and gel electrophoresis. Certainepitopes can also be identified by using large libraries of randompeptide sequences displayed on the surface of phage particles (phagelibraries). Alternatively, a defined library of overlapping peptidefragments can be tested for binding to the test antibody in simplebinding assays. In an additional example, mutagenesis of an antigenbinding domain, domain swapping experiments and alanine scanningmutagenesis can be performed to identify residues required, sufficient,and/or necessary for epitope binding. For example, domain swappingexperiments can be performed using a mutant of a target antigen in whichvarious fragments of the IL-20 polypeptide have been replaced (swapped)with sequences from a closely related, but antigenically distinctprotein (such as another member of the neurotrophin protein family). Byassessing binding of the antibody to the mutant IL-20, the importance ofthe particular antigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody binds to the same epitope as the other antibodies. Competitionassays are well known to those of skill in the art.

Other IL-20 Antagonists

IL-20 antagonists other than antibodies capable of interfering with theIL-20 signaling pathway as described above can be used in the methodsdescribed herein.

In some embodiments of the invention, the IL-20 antagonist comprises atleast one antisense nucleic acid molecule capable of blocking ordecreasing the expression of a functional IL-20 (e.g., a human IL-20) ora subunit of an IL-20 receptor (e.g., IL-20R1). Nucleotide sequences ofthe IL-20 and IL-20 receptor subunits are known and are readilyavailable from publicly available databases. See above disclosures. Itis routine to prepare antisense oligonucleotide molecules that willspecifically bind a target mRNA without cross-reacting with otherpolynucleotides. Exemplary sites of targeting include, but are notlimited to, the initiation codon, the 5′ regulatory regions, the codingsequence and the 3′ untranslated region. In some embodiments, theoligonucleotides are about 10 to 100 nucleotides in length, about 15 to50 nucleotides in length, about 18 to 25 nucleotides in length, or more.The oligonucleotides can comprise backbone modifications such as, forexample, phosphorothioate linkages, and 2′-0 sugar modifications wellknown in the art.

Alternatively, IL-20/IL-20R expression and/or release can be decreasedusing gene knockdown, morpholino oligonucleotides, small interfering RNA(siRNA or RNAi), microRNA or ribozymes, methods that are well-known inthe art. RNA interference (RNAi) is a process in which a dsRNA directshomologous sequence-specific degradation of messenger RNA. In mammaliancells, RNAi can be triggered by 21-nucleotide duplexes of smallinterfering RNA (siRNA) without activating the host interferon response.The dsRNA used in the methods disclosed herein can be a siRNA(containing two separate and complementary RNA chains) or a shorthairpin RNA (i.e., a RNA chain forming a tight hairpin structure), bothof which can be designed based on the sequence of the target gene.Alternatively, it can be a microRNA.

Optionally, a nucleic acid molecule to be used in the method describedherein (e.g., an antisense nucleic acid, a small interfering RNA, or amicroRNA) as described above contains non-naturally-occurringnucleobases, sugars, or covalent internucleoside linkages (backbones).Such a modified oligonucleotide confers desirable properties such asenhanced cellular uptake, improved affinity to the target nucleic acid,and increased in vivo stability.

In one example, the nucleic acid has a modified backbone, includingthose that retain a phosphorus atom (see, e.g., U.S. Pat. Nos.3,687,808; 4,469,863; 5,321,131; 5,399,676; and 5,625,050) and thosethat do not have a phosphorus atom (see, e.g., U.S. Pat. Nos. 5,034,506;5,166,315; and 5,792,608). Examples of phosphorus-containing modifiedbackbones include, but are not limited to, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkyl-phosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having 3′-5′ linkages, or 2′-5′ linkages. Suchbackbones also include those having inverted polarity, i.e., 3′ to 3′,5′ to 5′ or 2′ to 2′ linkage. Modified backbones that do not include aphosphorus atom are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. Such backbones include thosehaving morpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

In another example, the nucleic acid used in the disclosed methodsincludes one or more substituted sugar moieties. Such substituted sugarmoieties can include one of the following groups at their 2′ position:OH; F; O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl;O-alkynyl, S-alkynyl, N-alkynyl, and O-alkyl-O-alkyl. In these groups,the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ toC₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. They may also include attheir 2′ position heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an oligonucleotide, or a group for improving thepharmacodynamic properties of an oligonucleotide. Preferred substitutedsugar moieties include those having 2′-methoxyethoxy,2′-dimethylaminooxyethoxy, and 2′-dimethylaminoethoxyethoxy. See Martinet al., Helv. Chim. Acta, 1995, 78, 486-504.

In yet another example, the nucleic acid includes one or more modifiednative nucleobases (i.e., adenine, guanine, thymine, cytosine anduracil). Modified nucleobases include those described in U.S. Pat. No.3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering,pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, Englischet al., Angewandte Chemie, International Edition, 1991, 30, 613, andSanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289-302, CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the antisenseoligonucleotide to its target nucleic acid. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines(e.g., 2-aminopropyl-adenine, 5-propynyluracil and 5-propynylcytosine).See Sanghvi, et al., eds., Antisense Research and Applications, CRCPress, Boca Raton, 1993, pp. 276-278).

Any of the nucleic acids can be synthesized by methods known in the art.See, e.g., Caruthers et al., 1992, Methods in Enzymology 211, 3-19,Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al.,1997, Methods Mol. Bio. 74, 59, Brennan et al., 1998, BiotechnolBioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. It can also betranscribed from an expression vector and isolated using standardtechniques.

In other embodiments, the IL-20 antagonist comprises at least one IL-20or IL-20R inhibitory compound. As used herein, “IL-20 inhibitorycompound” or “IL-20R inhibitory compound” refers to a compound otherthan an anti-IL-20 or anti-IL-20R antibody that directly or indirectlyreduces, inhibits, neutralizes, or abolishes IL-20/IL-20R biologicalactivity. An IL-20/IL-20R inhibitory compound should exhibit any one ormore of the following characteristics: (a) binds to IL-20 or IL-20R andinhibits its biological activity and/or downstream pathways mediated byIL-20 signaling function; (b) prevents, ameliorates, or treats anyaspect of pancreatic cancer; (c) blocks or decreases IL-20 receptoractivation; (d) increases clearance of IL-20 or IL-20R; (e) inhibits(reduces) IL-20 or IL-20R synthesis, production or release. One skilledin the art can prepare other small molecules inhibitory compounds.

In some embodiments, an IL-20 or IL-20R inhibitory compound is an IL-20mutant, an IL-19 mutant, or an IL-24 mutant, which can bind to an IL-20receptor but cannot elicit signal transduction. Such a mutant may blockbinding of wild type IL-20 to an IL-20 receptor thus preventing IL-20signal transduction.

In other embodiments, the IL-20 or IL-20R inhibitory compounds describedherein are small molecules, which can have a molecular weight of aboutany of 100 to 20,000 daltons, 500 to 15,000 daltons, or 1000 to 10,000daltons. Libraries of small molecules are commercially available. Thesmall molecules can be administered using any means known in the art,including inhalation, intraperitoneally, intravenously, intramuscularly,subcutaneously, intrathecally, intraventricularly, orally, enterally,parenterally, intranasally, or dermally. In general, when theIL-20-antagonist according to the invention is a small molecule, it willbe administered at the rate of 0.1 to 300 mg/kg of the weight of thepatient divided into one to three or more doses. For an adult patient ofnormal weight, doses ranging from 1 mg to 5 g per dose can beadministered.

The above-mentioned small molecules can be obtained from compoundlibraries. The libraries can be spatially addressable parallel solidphase or solution phase libraries. See, e.g., Zuckermann et al. J. Med.Chem. 37, 2678-2685, 1994; and Lam Anticancer Drug Des. 12:145, 1997.Methods for the synthesis of compound libraries are well known in theart, e.g., DeWitt et al. PNAS USA 90:6909, 1993; Erb et al. PNAS USA91:11422, 1994; Zuckermann et al. J. Med. Chem. 37:2678, 1994; Cho etal. Science 261:1303, 1993; Carrell et al. Angew Chem. Int. Ed. Engl.33:2059, 1994; Carell et al. Angew Chem. Int. Ed. Engl. 33:2061, 1994;and Gallop et al. J. Med. Chem. 37:1233, 1994. Libraries of compoundsmay be presented in solution (e.g., Houghten Biotechniques 13:412-421,1992), or on beads (Lam Nature 354:82-84, 1991), chips (Fodor Nature364:555-556, 1993), bacteria (U.S. Pat. No. 5,223,409), spores (U.S.Pat. No. 5,223,409), plasmids (Cull et al. PNAS USA 89:1865-1869, 1992),or phages (Scott and Smith Science 249:386-390, 1990; Devlin Science249:404-406, 1990; Cwirla et al. PNAS USA 87:6378-6382, 1990; Felici J.Mol. Biol. 222:301-310, 1991; and U.S. Pat. No. 5,223,409).

In other embodiments, the IL-20 antagonists can be a polypeptidecomprising an extracellular portion of an IL-20 receptor (such as IL-20R1, IL-20R2, or IL-22R1), wherein the polypeptide specifically binds to11-20 and blocks its interaction with one or more IL-20 receptors. Insome embodiments, the extracellular portion of the IL-20 receptor isfused to a Fc domain of antibody. Examples of the soluble receptors aredescribed in PCT WO 01/46232.

Identification of IL-20 Antagonists

IL-20 antagonists can be identified or characterized using methods knownin the art, whereby reduction, amelioration, or neutralization of anIL-20 biological activity is detected and/or measured. For example, anELISA-type assay may be suitable for qualitative or quantitativemeasurement of IL-20 mediated kinase activation by measuring thephosphorylation of proteins activated through an IL-20 cascade. Examplesinclude JNK, ERK, AKT, p38, STATS and TRAF6.

The IL-20 antagonists can also be identified by incubating a candidateagent with IL-20 or IL-20R and monitoring any one or more of thefollowing characteristics: (a) binding to IL-20 or IL-20R and inhibitingits biological activity and/or downstream pathways mediated by IL-20signaling function; (b) preventing, ameliorating, or treating any aspectof pancreatic cancer; (c) blocking or decreasing IL-20 receptoractivation; (d) increasing clearance of IL-20 or IL-20R; (e) inhibiting(reducing) IL-20 synthesis, production or release. In some embodiments,an IL-20 antagonist is identified by incubating a candidate agent withIL-20 or IL-20R and monitoring binding and attendant reduction orneutralization of a biological activity of IL-20 or IL-20R. The bindingassay may be performed with purified IL-20 or IL-20R polypeptide(s), orwith cells naturally expressing, or transfected to express, IL-20 orIL-20R polypeptide(s). In one embodiment, the binding assay is acompetitive binding assay, where the ability of a candidate antibody tocompete with a known IL-20 antagonist for IL-20 or IL-20R binding isevaluated. The assay may be performed in various formats, including theELISA format. In other embodiments, an IL-20 antagonist is identified byincubating a candidate agent with IL-20 or IL-20R (e.g., IL-20R1) andmonitoring attendant inhibition of IL-20R1/IL-20R2 complex formation orIL-20R2/IL-22R1 complex formation. Following initial identification, theactivity of a candidate IL-20 antagonist can be further confirmed andrefined by bioassays, known to test the targeted biological activities.Alternatively, bioassays can be used to screen candidates directly.

The examples provided below provide a number of assays that can be usedto screen candidate IL-20 antagonists. Bioassays include but are notlimited to flow cytometry of determine competitive binding of IL-20 tocells in the presence of candidate IL-20 antagonists; and inhibition ofIL-20-induced apoptosis in renal epithelial cells. In addition, RT-PCRor Real-time PCR which can be used to directly measure IL-20 expressionor to measure expression of genes upregulated by IL-20 such as TNFαMCP-1, IL-1β, IL-6 and VEGF.

Pharmaceutical Compositions

One or more of the above-described IL-20 antagonist can be mixed with apharmaceutically acceptable carrier (excipient), including buffer, toform a pharmaceutical composition for use in alleviating pancreaticcancer. “Acceptable” means that the carrier must be compatible with theactive ingredient of the composition (and preferably, capable ofstabilizing the active ingredient) and not deleterious to the subject tobe treated. Pharmaceutically acceptable excipients (carriers) includingbuffers, which are well known in the art. See, e.g., Remington: TheScience and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams andWilkins, Ed. K. E. Hoover. In one example, a pharmaceutical compositiondescribed herein contains more than one anti-IL-20 or anti-IL-20Rantibodies that recognize different epitopes of the target antigen. Inanother example, the pharmaceutical composition comprises at least twodifferent-typed IL-20 antagonists (e.g., one antibody and one smallmolecule).

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueoussolutions. (Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations used, and may comprise buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

In some examples, the pharmaceutical composition described hereincomprises liposomes containing the IL-20 antagonist (such as anantibody), which can be prepared by methods known in the art, such asdescribed in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985);Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat.Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomescan be generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The active ingredients (e.g., an IL-20 antagonist) may also be entrappedin microcapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are known in theart, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an IL-20antagonist with Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Use of IL-20 Antagonists for Treating Pancreatic Cancer

To practice the method disclosed herein, an effective amount of thepharmaceutical composition described above can be administered to asubject (e.g., a human) in need of the treatment via a suitable route,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, IL-20 antagonists can be aerosolizedusing a fluorocarbon formulation and a metered dose inhaler, or inhaledas a lyophilized and milled powder.

The subject to be treated by the methods described herein can be amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, horses, dogs, cats,mice and rats. A human subject who needs the treatment may be a humanpatient having, at risk for, or suspected of having pancreatic cancer(e.g., pancreatic ductal adenocarcinoma, exocrine pancreatic cancer,non-adenocarcinoma pancreatic cancer, pancreatic adenocarcinoma,pancreatic cystic cancer, acinar cell cancer, endocrine pancreaticcancer, gastrinomas, insulinomas, somatostatinomas, VlPomas,glucagonomas, pancreatoblastoma, pancreatic sarcoma or lymphoma). Asubject having pancreatic cancer can be identified by routine medicalexamination, e.g., laboratory tests, pancreatic functional tests,pancreas biopsy, CT scans, ultrasounds, or endoscopic retrogradecholangio pancreatography (ERCP). A subject suspected of havingpancreatic cancer might show one or more symptoms of the disorder, e.g.,stomach pain, back pain, jaundice, weight loss, diabetes, itching,sickness, bowel changes, fever and shivering, indigestion, or bloodclots. A subject at risk for pancreatic cancer can be a subject havingone or more of the risk factors for that disorder. For example, riskfactors associated with pancreatic cancer include (a) hereditarypancreatitis, (b) age (pancreatic cancer is more frequent in people over40), and (c) family history of pancreatic cancer.

“An effective amount” as used herein refers to the amount of each activeagent required to confer therapeutic effect on the subject, either aloneor in combination with one or more other active agents. Effectiveamounts vary, as recognized by those skilled in the art, depending onthe particular condition being treated, the severity of the condition,the individual patient parameters including age, physical condition,size, gender and weight, the duration of the treatment, the nature ofconcurrent therapy (if any), the specific route of administration andlike factors within the knowledge and expertise of the healthpractitioner. These factors are well known to those of ordinary skill inthe art and can be addressed with no more than routine experimentation.It is generally preferred that a maximum dose of the individualcomponents or combinations thereof be used, that is, the highest safedose according to sound medical judgment. It will be understood by thoseof ordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of pancreatic cancer. Alternatively, sustained continuousrelease formulations of an IL-20 antagonist may be appropriate. Variousformulations and devices for achieving sustained release are known inthe art.

In one example, dosages for an IL-20 antagonist as described herein maybe determined empirically in individuals who have been given one or moreadministration(s) of IL-20 antagonist. Individuals are given incrementaldosages of the antagonist. To assess efficacy of the antagonist, anindicator of pancreas function (such as levels of elastase) can befollowed.

Generally, for administration of any of the antibodies described herein,an initial candidate dosage can be about 2 mg/kg. For the purpose of thepresent disclosure, a typical daily dosage might range from about any of0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate pancreatic cancer, or a symptom thereof. An exemplary dosingregimen comprises administering an initial dose of about 2 mg/kg,followed by a weekly maintenance dose of about 1 mg/kg of the antibody,or followed by a maintenance dose of about 1 mg/kg every other week.However, other dosage regimens may be useful, depending on the patternof pharmacokinetic decay that the practitioner wishes to achieve. Forexample, dosing from one-four times a week is contemplated. In someembodiments, dosing ranging from about 3 μg/mg to about 2 mg/kg (such asabout 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays. The dosing regimen(including the antibody used) can vary over time.

When the IL-20 antagonist is not an antibody, it may be administered atthe rate of about 0.1 to 300 mg/kg of the weight of the patient dividedinto one to three doses, or as disclosed herein. In some embodiments,for an adult patient of normal weight, doses ranging from about 0.3 to5.00 mg/kg may be administered. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history, as well as the properties of theindividual agents (such as the half-life of the agent, and otherconsiderations well known in the art).

For the purpose of the present disclosure, the appropriate dosage of anIL-20 antagonist will depend on the specific IL-20 antagonist(s) (orcompositions thereof) employed, the type and severity of pancreaticcancer, whether the antagonist is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antagonist, and the discretion of the attendingphysician. Typically the clinician will administer an IL-20 antagonist,such as an anti-IL-20 or anti-IL-20R antibody, until a dosage is reachedthat achieves the desired result. Administration of an IL-20 antagonistcan be continuous or intermittent, depending, for example, upon therecipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of an IL-20 antagonist (forexample if the IL-20 antagonist is an anti-IL-20 antibody) may beessentially continuous over a preselected period of time or may be in aseries of spaced dose, e.g., either before, during, or after developingpancreatic cancer.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has pancreatic cancer, a symptom of pancreatic cancer, or apredisposition toward the disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisorder, the symptom of the disease, or the predisposition toward thedisease.

Alleviating pancreatic cancer includes delaying the development orprogression of the disease, or reducing disease severity. Alleviatingthe disease does not necessarily require curative results. As usedtherein, “delaying” the development of a disease (such as pancreaticcancer) means to defer, hinder, slow, retard, stabilize, and/or postponeprogression of the disease. This delay can be of varying lengths oftime, depending on the history of the disease and/or individuals beingtreated. A method that “delays” or alleviates the development of adisease, or delays the onset of the disease, is a method that reducesprobability of developing one or more symptoms of the disease in a giventime frame and/or reduces extent of the symptoms in a given time frame,when compared to not using the method. Such comparisons are typicallybased on clinical studies, using a number of subjects sufficient to givea statistically significant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of pancreatic cancer includes initial onsetand/or recurrence.

In some embodiments, the IL-20 antagonist (e.g., an anti-IL-20 antibodyor anti-IL-20R antibody such as anti-IL-20R1 antibody) described hereinis administered to a subject in need of the treatment at an amountsufficient to reduce the level of the IL-20 receptor/IL-20-mediatedsignaling by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater). In other embodiments, the antagonist is administered in anamount effective in reducing at least on symptom of pancreatic cancer.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. Inaddition, it can be administered to the subject via injectable depotroutes of administration such as using 1-, 3-, or 6-month depotinjectable or biodegradable materials and methods.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In one embodiment, an IL-20 antagonist is administered via site-specificor targeted local delivery techniques. Examples of site-specific ortargeted local delivery techniques include various implantable depotsources of the IL-20 antagonist or local delivery catheters, such asinfusion catheters, an indwelling catheter, or a needle catheter,synthetic grafts, adventitial wraps, shunts and stents or otherimplantable devices, site specific carriers, direct injection, or directapplication. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat.No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, expression vector, or subgenomic polynucleotides canalso be used. Receptor-mediated DNA delivery techniques are describedin, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiouet al., Gene Therapeutics: Methods And Applications Of Direct GeneTransfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988)263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc.Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991)266:338. Therapeutic compositions containing a polynucleotide areadministered in a range of about 100 ng to about 200 mg of DNA for localadministration in a gene therapy protocol. In some embodiments,concentration ranges of about 500 ng to about 50 mg, about 1 μg to about2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNAor more can also be used during a gene therapy protocol.

The therapeutic polynucleotides and polypeptides described herein can bedelivered using gene delivery vehicles. The gene delivery vehicle can beof viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy(1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).Expression of such coding sequences can be induced using endogenousmammalian or heterologous promoters and/or enhancers. Expression of thecoding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell. Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

It is also apparent that an expression vector can be used to directexpression of any of the protein-based IL-20 antagonists describedherein (e.g., anti-IL-20 antibody, or anti-IL-20R antibody). Forexample, other IL-20 receptor fragments that are capable of blocking(from partial to complete blocking) IL-20 and/or an IL-20 biologicalactivity are known in the art.

The particular dosage regimen, i.e., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history.

In some embodiments, more than one IL-20 antagonist, such as an antibodyand a small molecule IL-20 inhibitory compound, may be administered to asubject in need of the treatment. The antagonist can be the same type ordifferent from each other. At least one, at least two, at least three,at least four, at least five different IL-20 antagonists can beco-administered. Generally, those IL-20 antagonists have complementaryactivities that do not adversely affect each other. IL-20 antagonistscan also be used in conjunction with other agents that serve to enhanceand/or complement the effectiveness of the agents.

Treatment efficacy can be assessed by methods well-known in the art.

Kits for Use in Treating Pancreatic Cancer

The present disclosure also provides kits for use in alleviatingpancreatic cancer. Such kits can include one or more containerscomprising an IL-20 antagonist (such as an antibody, e.g., mAb7E or itsfunctional variant, mAb7GW or its functional variant, or mAb51D or itsfunctional variant). In some embodiments, the IL-20 antagonist is anyantibody capable of interfering with the IL-20 signaling pathway asdescribed herein. In other embodiments, the kit comprises an IL-20antagonist that is other than the just-noted antibody.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of the IL-20antagonist to treat, delay the onset, or alleviate pancreatic canceraccording to any of the methods described herein. The kit may furthercomprise a description of selecting an individual suitable for treatmentbased on identifying whether that individual has pancreatic cancer. Instill other embodiments, the instructions comprise a description ofadministering an IL-20 antagonist to an individual at risk of pancreaticcancer.

The instructions relating to the use of an IL-20 antagonist generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, delaying the onset and/or alleviating pancreatic cancer.Instructions may be provided for practicing any of the methods describedherein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an IL-20 antagonist, such as an anti-IL-20 antibody.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the invention provides articles of manufacture comprisingcontents of the kits described above.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Example 1: Inhibitory Effects of an Anti-IL-20 Antibody on Tumor Growth

The effect of IL-20 inhibition on tumor growth was analyzed in a mousemodel of pancreatic cancer. Six-week-old male nude mice (BALB/c Nude)mice were used in the experiment. The left mammary fat pad of each mousewas subcutaneously injected with BxPC-3 cells (2×10⁶). The successfulrate for subcutaneous (s.c.) tumor implantation was 100%. The mice werethen randomly assigned to 3 groups (n=4 in each group), and treated withphosphate-buffered saline (PBS), an anti-IL-20 monoclonal antibody,mAb7E (6 mg/kg; s.c.), or mouse immunoglobulin G (mIgG; 6 mg/kg; s.c.)two times per week for the duration of the treatment regimen. Theantibody was injected (s.c.) into the periphery of the growing tumor intumor-bearing nude mice. Healthy controls were not injected with tumorcells. The tumor size was measured with a caliper in three perpendiculardimensions and calculated using the following formula: tumorsize=length×width×depth. Forty eight days after the tumor cells had beeninjected, the mice were killed using CO₂, and the tumor tissue washarvested and weighed.

Tumors harvested from mice treated with mAb7E have significantly smallersizes than tumors harvested from mice treated with PBS or mIgG (FIG. 1).The differences in tumor sizes can easily be visually observed (FIG. 2,panel A). Accordingly, tumor mass harvested from mice treated with mAb7Eweight was significantly less than tumor mass harvested from micetreated with PBS or mIgG (FIG. 2, panel B).

The above-results clearly show that anti-IL-20 antibody significantlyreduced tumor growth as measured by tumor size and tumor weight,indicating that anti-IL-20 therapies would be effective in treatingpancreatic cancer.

Example 2: Treatment with mAb7E Prolongs Survival Rates in Mice HavingKPC-Induced Pancreatic Cancer

The effect of IL-20 inhibition on survival rates in a mouse model ofpancreatic ductal adenocarcinoma (the KPC mouse model) was analyzed. KPCmice develop pancreatic cancer when they reach 4 weeks old. Olive etal., Clin. Cancer Res. 12(18):5277-5287 (2006) and Olive et al.,Science, 324(5933):1457-1461 (2009). Twenty-five days after birth, theKPC mice were randomly divided to 3 groups (n=10 in each group), andtreated with phosphate-buffered saline (PBS), 7E (mAb7E, 6 mg/kg; i.p.),or mouse immunoglobulin G (mIgG; 6 mg/kg; i.p.) two times per week untilthe mouse dies. Mice treated with PBS or mIgG died at day 55-60 (FIG.3). Survival rates of KPC mice were significantly prolonged by treatmentwith mAb7E (FIG. 3).

The above-results clearly show that mAb7E significantly prolonged thesurvival rate of mice having pancreatic ductal adenocarcinoma,indicating that anti-IL-20 therapies are effective in alleviatingpancreatic cancer and treating pancreatic cancer.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS AND SCOPE

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

What is claimed is:
 1. A method for treating or delaying the onset ofpancreatic cancer in a subject, comprising administering to a subject inneed thereof an effective amount of a pharmaceutical compositioncomprising an IL-20 antagonist.
 2. The method of claim 1, wherein theIL-20 antagonist is an antibody that binds either IL-20 or an IL-20receptor, thereby inhibiting a signaling pathway mediated by IL-20. 3.The method of claim 2, wherein the antibody is an antibody that bindshuman IL-20.
 4. The method of claim 3, wherein the antibody is afull-length antibody or an antigen-binding fragment thereof.
 5. Themethod of claim 4, wherein the antibody is a human antibody, a humanizedantibody, a chimeric antibody, or a single-chain antibody.
 6. The methodof claim 3, wherein the anti-IL-20 antibody is monoclonal antibodymAb7E, an antigen-binding fragment thereof, or a functional variantthereof.
 7. The method of claim 6, wherein the functional variantcomprises the same complementary determining regions (CDRs) as mAb7E. 8.The method of claim 7, wherein the functional variant is a humanizedantibody of mAb7E.
 9. The method of claim 8, wherein the humanizedantibody comprises a heavy chain variable region (V_(H)), whichcomprises the amino acid sequence of SEQ ID NO:8, and a light chainvariable region (V_(L)), which comprises the amino acid sequence of SEQID NO:12 or SEQ ID NO:13.
 10. The method of claim 2, wherein theantibody is an antibody that binds a human IL-20 receptor.
 11. Themethod of claim 10, wherein the antibody binds subunit R1 of the humanIL-20 receptor.
 12. The method of claim 11, wherein the antibody is afull-length antibody or an antigen-binding fragment thereof.
 13. Themethod of claim 11, wherein the antibody is a human antibody, ahumanized antibody, a chimeric antibody, or a single-chain antibody. 14.The method of claim 11, wherein the antibody that binds subunit R1 ofthe human IL-20 receptor is an antibody comprising the same V_(H) andV_(L) chain as monoclonal antibody mAb51D or mAb7GW, or a functionalvariant thereof.
 15. The method of claim 14, wherein the functionalvariant comprises the same complementary determining regions (CDRs) asmAb51D or mAb7GW.
 16. The method of claim 14, wherein the functionalvariant is a humanized antibody of mAb51D or mAb7GW.
 17. The method ofclaim 1, wherein the subject is a human patient having or beingsuspected of having pancreatic cancer.